Characterization of hemolytic toxins from periodontopathic genus Prevotella and their clinically roles

2002 Fiscal Year Final Research Report Summary

• Project/Area Number: 13672197
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Periodontal dentistry
• Research Institution: Nihon University
• Principal Investigator: TAKADA Kazuko School of Dentistry at Matsudo, Lecturer (Full-Time), 松戸歯学部, 講師 (20120496)
• Co-Investigator(Kenkyū-buntansha): HIRASAWA Masatomo School of Dentistry at Matsudo, Professor, 松戸歯学部, 教授 (60095453)
• Project Period (FY): 2001 – 2002
• Keywords: Periodontitis / Prevotella intermedia / Black-pigmented Gram negative rods / Hemolytic toxin / Reductant

Research Abstract

Black-pigmented anaerobic Gram-negative rods such as Porphyromonas gingivalis and Prevotella spp. Have been implicated as key pathogenic and causative agents of adult periodontitis. Moreover Prevotella spp. Has been frequently recovered from subgingival plaque in patients suffering from acute necrotizing gingivitis and pregnancy gingivitis in addition to adult periodontitis, and is thus suggested to have pathogenic roles in these conditions. In this study isolation and characterization of extracellular hemolysin from P. intermedia were examined.
The hemolysin from P. intermedia has been purified from culture supernatant and characterized. The hemolysin produced clear β-hemolytic zone on a blood agar plate. Hemolytic activity was 2.5-fold greater in culture supernatant compared to that cell-associated. The isolation and purification procedure involved ammonium sulfate and polyethylene glycol precipitations and ion-exchange chromatographies on DEAE-Sephacel and CM-Sepharose. The optimal p H of the hemolysin was 7.5. Hemolytic activity was stimulated by reductants such as cysteine, dithiothreitol and glutathione etc., and was lost upon oxidation Trypsin or heat treatment resulted in complete inhibition of hemolytic activity. Cation and EDTA did not affect the activity. These results indicate that the β-hemolysin from P. intermedia, appeared to belong to a family of toxins known as the thiol-activated toxin.
The other hemolytic substance was extracted and separated from culture supernatant by ammonium sulfate fractionation, Biogel P6 and P2 gel filtration column chromatographies. The molecular weight of the purified substance was estimated 1300 by Biogel P2 column. The range of pH 6.0-7.5 was obatained the stable hemolytic activity. The hemolytic substance showed 0.5 Rf value on thin layer chromatography (TLC) using chloroform : methanol : water (65:35:5) by ninhydrin as a developing solvent. Cation and EDTA did not affect the hemolytic substance The hemolytic substance is likely δ hemolysin, which was produced by Staphylococcus aureus and Streptococcus pyogenes. It is possible that β- and δ- hemolysin produced by P. intermedia contributed to this organism's pathogenicity.